Compositions comprising magnolia, phellodendron, theanine and/or whey protein

ABSTRACT

An anxiolytic composition includes a synergistic combination of one or more of an extract of magnolia, an extract of phellodendron, an L-theanine, a whey protein composition, and/or an s-adenosylmethionine (SAMe).

This application is a continuation patent application claiming priorityto International Application No. PCT/US2016/049630 filed on Aug. 31,2016, which in turn claims priority to U.S. Provisional PatentApplication Ser. No. 62/212,080 filed on Aug. 31, 2015, the entirety ofthe contents of each of which are incorporated by reference herein.

TECHNICAL FIELD

The present application relates generally to anxiolytic compositionscontaining magnolia, phellodendron, theanine, a whey protein, and/ors-adenosylmethionine.

BACKGROUND

Anxiety, fear, and stress play major roles or contribute in some way tomany human and non-human animal behavioral disorders. For example, withregard to canines and felines, storm phobias, noise phobias, socialavoidance, fear-related aggression, compulsive disorders, and submissiveurination have obvious anxiety components, but even problems such asurine marking, territorial aggression, and resource guarding can befueled by fear or anxiety. Fear and anxiety disorders affect anestimated 23 million dogs in the US at a cost of more than $1 billion intreatment and property damage. Of dogs relinquished to shelters, perhaps40% or more are abandoned due to behavioral issues, while 14% of catsare surrendered due to behavioral problems. In pet cats, behaviorproblems are still the most common cause of euthanasia. In a recentsurvey of pet owners, 41% of dog owners reported that they have owned adog with anxiety issues at some time, with 29% of currently-owned dogsbeing affected. The most prevalent types of anxiety reported by theseowners included noise phobia (17%), separation anxiety (13%), andgeneralized anxiety (5%).

Anxiety in horses is similarly a common problem in the equine industry,contributing to behavioral problems, training issues and poorperformance. In some cases, increased levels of anxiety are associatedwith health related issues such as gastric ulcers, which have been shownin some studies to impact a large percentage of horses. Manifestationsof anxiety in horses is commonly seen not only in the form of gastriculcers, but also is expressed behaviorally in the form of bolting,jumping, panicking, trailering difficulties, pacing, stall weaving,pawing, and biting. The most common risk factor for anxiety disorders indogs appears to be acquisition from animal shelters or multiple fosterhomes, with up to 68.3% of dogs adopted from shelters exhibiting sometype of anxiety disorder. One retrospective study on behavior diagnosesindicates possible breed predilections in Dalmatians, English springerspaniels, German shepherd dogs, and mixed breed dogs, while anotherstudy suggests cocker spaniels, schnauzers, and dachshunds may be atrisk for developing separation anxiety. The incidence of anxietydisorders does appear to increase with age, most likely due to a loss ofcognitive function, from an incidence of 22.5% in dogs less than 3 yearsof age to an incidence of 36.5% in dogs 8 years of age and over.

Fear is an emotional response due to the presence of a specific stimulus(object, noise, individual, etc.) that the pet perceives as a threat ora danger. In contrast, an anxiety is a reaction of apprehension oruneasiness to an anticipated danger or threat. Anxiety, therefore, maybe displayed in the absence of an identifiable stimulus, whereas withfear, a stimulus can usually be identified. While anxiety and theaccompanying physiological stress are unlikely to be pathologic in theshort-term, when stress and anxiety become a chronic state, the health,welfare, and lifespan of the pet may become compromised. Therefore, toprovide optimum health and welfare for all pets, veterinary healthcarepractitioners consider anxiety as an additional possible disease statein companion animals. At the extreme end of the spectrum, a phobia is aprofound, excessive, abnormal fear response that occurs without thepresence of a true threat or is out of proportion to the needs fordealing with an actual threat. While fears may be normal adaptiveresponses, phobias are abnormal, maladaptive, and typically interferewith normal function. Anxiety, which inhabits the middle of thespectrum, is often overlooked or misunderstood, but may have asignificant impact on a pet's daily well-being.

Clinical signs of fear and anxiety in dogs include hypervigilance,elimination, destruction, excessive vocalization, hyper-salivation,panting, hiding, trembling, and escape behaviors. In cats, chronicanxiety and fear can also lead to secondary behavioral problems such asover-grooming, spraying, and inter-cat aggression, and can predisposethe cat to health problems owing to a compromised immune system. Sincemost of the clinical signs of fear and anxiety are destructive anddistressing to both pet and owner, one can easily understand why petowners would be likely to seek out advice for such disorders. Notcoincidentally, the most common risk factors for relinquishment toanimal shelters and euthanasia for both dogs and cats includehouse-soiling, destruction, aggression, and hyperactive behavior—allpotential clinical signs of anxiety.

Anxiety disorders rarely occur alone, and often occur in combination.Thunderstorm and noise phobias do not necessarily occur simultaneously;however, thunderstorm phobia, noise phobia, and separation anxiety occursignificantly more often together than would be expected were theseconditions independent. Such evidence suggests that the precise cause ofa pet's anxiety could be difficult to isolate, and multiple pathologiescould be occurring in the same pet.

Although the “triggers” and manifestation of behavioral disorders inhumans may be different, anxiety, fear, and stress contribute to thehuman disorders. In an effort to alleviate the behavior disorders ofhuman and non-human animals, certain synthetic drugs have beendeveloped. For instance, clomipramine hydrochloride, fluoxetinehydrochloride, benzodiazepine, and acepromazine maleate are all drugcompositions that have been used in an attempt to alleviate anxiety inhumans and/or non-human animals. While these drugs may provide somerelief from anxiety, a significant downside of these drugs is that theycan be sedating, and the human or non-human animal experiences lethargyor sleepiness. Moreover, many of these drugs are synthetic compositions,which humans may be reluctant to ingest or administer to their pets.

The biochemistry of anxiety is extremely complicated and, to a greatextent, still poorly understood. Studies have shown that nearly everytype of neurotransmitter and hormone, from serotonin, γ-aminobutyricacid (GABA), glutamate, and dopamine to cortisol, adrenaline, and eventhyroid hormone can play some role in anxiety. Anxiety, in many ways, issimply the biochemical reaction to brain stress. When something causesany change to the delicate chemical balance in the brain, anxiety isoften the result.

The ideal management for anxiety, therefore, should be multi-modal,increasing the likelihood that one or more of the mechanisms of actionwill target and correct any given underlying chemical imbalance. CommonFDA-approved pharmaceuticals such as serotonin-selective re-uptakeinhibitors (SSRI's), tricyclic antidepressants (TCA's), andbenzodiazepines, however, typically work via a single mechanism ofaction.

Due to the difficulty in tracking adverse drug reactions in combinationdrugs, the FDA has become increasingly reluctant to approve drugs withmultiple active ingredients. Therefore, a multi-modal approach usingpharmaceuticals alone is unlikely. Nutritional and herbal supplements,which are regulated more like foods than drugs by the FDA, represent anovel delivery method for multiple ingredients, offering a much greateropportunity for the multi-modal management of anxiety. For this andother reasons, the use of natural products for human and non-humananimals is becoming increasingly popular as consumers seek alternativesto pharmaceuticals. Some of these natural products are beingincorporated into dietary supplements and various foods.

In light of the discussion above, the need exists for a natural productcomposition that has anxiolytic properties. This composition should helpsupport normal behavior and facilitate a calming effect, while notcausing extreme lethargy or sleepiness. In turn, such compositionsshould work synergistically to manage and control the clinical signs orsymptoms of anxiety.

SUMMARY

In accordance with the purposes and benefits described herein, in oneaspect of the present disclosure an anxiolytic composition is providedcomprising a synergistic combination of L-theanine and a whey protein.In embodiments, the whey protein comprises alpha-lactalbumin. Theanxiolytic composition provides an anxiolytic change in a releasepattern of one or more neurotransmitters including γ-aminobutyric acid(GABA) and serotonin. The composition may be formulated for oraladministration to a mammal, including a human, a companion animal, andan equine animal.

In embodiments, the synergistic combination further comprises an extractof magnolia and an extract of phellodendron. In embodiments, thesynergistic combination further comprises RELORA.

In another aspect, a method is described for reducing, ameliorating, ortreating symptoms of anxiety, comprising administration of a synergisticcombination of L-theanine and a whey protein to a mammal. Inembodiments, the synergistic combination further comprises an extract ofmagnolia and an extract of phellodendron. In embodiments, the wheyprotein is provided as a whey protein concentrate comprisingalpha-lactalbumin. The method further includes orally administering thesynergistic combination in an amount sufficient to provide an anxiolyticchange in a release pattern of one or more neurotransmitters, includingin an embodiment γ-aminobutyric acid (GABA) and serotonin. The mammalmay be a human, a canine, a feline, or an equine.

In yet another aspect, an anxiolytic composition is provided comprisinga synergistic combination of L-theanine and s-adenosylmethionine (SAMe).The synergistic combination provides an anxiolytic change in a releasepattern of one or more neurotransmitters including glutamate andγ-aminobutyric acid (GABA). The composition may be formulated for oraladministration to a mammal. In embodiments, the SAMe comprises a SAMephytate salt.

In still yet another aspect, an anxiolytic composition is providedcomprising a synergistic combination of L-theanine, a whey protein, andRELORA. In embodiments, the whey protein comprises alpha-lactalbumin. Inembodiments, the whey protein is a whey protein concentrate.

In embodiments, the composition comprises at least 2.0 mg of L-theanine,at least 0.5 mg of whey protein, and at least 0.5 mg of RELORA. In otherembodiments, the composition comprises at least 2.0 mg of L-theanine, atleast 0.5 mg of whey protein, and at least 3.0 mg of RELORA.

In the following description, there are shown and described severalpreferred embodiments of the disclosed anxiolytic compositions andassociated methods. As it should be realized, the compositions andmethods are capable of other, different embodiments and their severaldetails are capable of modification in various, obvious aspects allwithout departing from the compositions and methods as set forth anddescribed in the following claims. Accordingly, the drawings anddescriptions should be regarded as illustrative in nature and not asrestrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawing figures incorporated herein and forming a partof the specification, illustrate several aspects of the disclosedanxiolytic compositions, and together with the description serve toexplain certain principles thereof. In the drawing:

FIG. 1 illustrates basal serotonin (5-HT) output followingadministration of compositions according to the present disclosure;

FIG. 2 illustrates basal GABA output following administration ofcompositions according to the present disclosure;

FIG. 3 illustrates basal glutamate output following administration ofcompositions according to the present disclosure;

FIG. 4 illustrates a pairwise comparison of GABA output followingadministration of an embodiment of a composition according to thepresent disclosure;

FIG. 5 illustrates a pairwise comparison of glutamate output followingadministration of the embodiment of FIG. 4;

FIG. 6A illustrates results of an Elevated-Plus Maze (EPM) testfollowing administration of compositions according to the presentdisclosure;

FIG. 6B illustrates a pairwise comparison of the EPM test followingadministration of an embodiment of a composition according to thepresent disclosure;

FIG. 6C illustrates a pairwise comparison of the EMP test followingadministration of another embodiment of a composition according to thepresent disclosure;

FIG. 7A illustrates results of an Open Field (OF) test followingadministration of compositions according to the present disclosure;

FIG. 7B illustrates a pairwise comparison of the OF test followingadministration of an embodiment of a composition according to thepresent disclosure; and

FIG. 7C illustrates a pairwise comparison of the OF test followingadministration of another embodiment of a composition according to thepresent disclosure.

Reference will now be made in detail to embodiments of the disclosedanxiolytic compositions, examples of which are illustrated in theaccompanying drawing figures.

DETAILED DESCRIPTION

The present invention relates to one or more compositions of magnolia,phellodendron, theanine, s-adenosylmethionine (SAMe), and/or wheyprotein that may be used by any human or non-human animal. In oneembodiment, the composition includes Magnolia officinalis, Phellodendronamurense, L-theanine, and alpha-lactalbumin. This combination ofmaterials may involve a number of different biochemical processes.Accordingly, the composition provides a multimodal approach toalleviating anxiety. Without intending to be bound by any particulartheory, one potential mode is by acting on multiple neurotransmitters(such as glutamate or GABA), increased production of neurotransmitterlevels such as serotonin, or by altering brain wave activity.

As further background, Magnolia officinalis is a species of Magnolianative to China. The bark and/or extract of the Magnolia officinalisinclude honokiol and magnolol, which enhance the activity of bothsynaptic and extra-synaptic GABA receptors in the brain. GABA is thebrain's chief inhibitory neurotransmitter which modulates the activityof overexcited neurons stimulated by fear and anxiety. The action ofthese compounds is thought to be selective binding to specific GABAreceptors which may explain why the effects of honokiol and magnolol areanxiolytic, without causing sedation.

Phellodendron amurense is a species of tree commonly called the Amurcork tree. Phellodendron fruit, bark and/or extracts are rich in thecompound berberine. The combination of Magnolia plus Phellodendronextracts is synergistic, with the combination controlling stress andanxiety more effectively than either compound used alone. Generally,synergy refers to the effect wherein a combination of two or morecomponents provides a result which is greater than the sum of theeffects produced by the agents when used alone. In preferred embodimentsof the present invention, the synergistic effect is greater than anadditive effect. The synergism observed with Magnolia plus Phellodendronextracts may be due to the fact that berberine inhibits the release ofglutamate by pre-synaptic neurons into the synaptic cleft. Theexcitatory neurotransmitter glutamate and the inhibitoryneurotransmitter GABA are both modulated at the synaptic level by thecombination. In a laboratory model, the combination of Magnolia andPhellodendron reduced anxiety in beagles in a placebo-controlledclinical trial of noise-induced anxiety. In some embodiments of thepresent invention, the combination of Magnolia and Phellodendron may beadministered as a commercially available preparation (RELORA;InterHealth Nutraceuticals, Inc., Benicia, Calif.).

The neurotransmitters glutamate and GABA work antagonistically toregulate interactions among neurons in the brain. Glutamate is theprimary excitatory neurotransmitter, making neurons more susceptible tostimuli and electrical stimulation. Glutamate plays a significant rolein fear and anxiety, which are often the result from over-stimulatedneurons. GABA is the primary inhibitory neurotransmitter in the nervoussystem and plays an equally essential role in controlling anxiety bydampening and/or reversing the effects of glutamate.

In one embodiment, the combination of Magnolia plus Phellodendronextracts is synergistic, with the combination controlling stress andanxiety more effectively than either compound used alone. This synergismis due to the effects these compounds have on both glutamate and GABA atthe synaptic level. Magnolia officinalis extracts, more specifically theconstituents honokiol and magnolol, enhance the activity of bothsynaptic and extra-synaptic GABA receptors in the brain. Phellodendronamurense extracts contain berberine. Berberine inhibits the release ofglutamate by pre-synaptic neurons into the synaptic cleft. Magnoliaextracts, therefore, enhance the effects of stabilizing GABA whileberberine from Phellodendron extracts blocks the release of excitatoryglutamate.

Berberine is a bright yellow ammonium salt found in Phellodendronamurense, and can also be found in such plants as the Oregon grape,barberry, goldenseal, goldenthread, and tree turmeric. Berberine isusually found in the roots, rhizomes, stems, and bark.

L-theanine is a structural analogue of the amino acid glutamate, themost important excitatory neurotransmitter of the nervous system.Theanine, found naturally in many types of tea, is thought to exertneuro-protective effects by binding and blocking glutamate receptors,thus reducing excitatory impulses and lowering the stimulatory effectsof glutamate. Theanine increases the levels of stabilizingneurotransmitters such as serotonin, dopamine, and GABA in the brain.Theanine also directly stimulates the production of alpha brain waves,which create a state of deep relaxation, wakefulness, and mentalalertness.

Alpha-lactalbumin is a component of certain whey protein compositionsand a high-quality protein source which supplements amino acids inanimals. Milk has long been considered a beverage with post-prandialcalming properties, especially in infants and young animals. The firsthuman studies on the anxiolytic effects of milk originated in the 1930'sand confirmed the calming effects of certain proteins in milk.Alpha-lactalbumin exerts neuro-protective properties by providing aminoacid precursors to the antioxidant glutathione (cysteine) and themood-enhancing neurotransmitter serotonin (tryptophan).

Since the brain consumes approximately 20% of the oxygen utilized by thebody, reactive oxygen species (ROS) are generated at extremely highrates, and brain cells are especially prone to oxidative damage. A lossof neurons in the mature brain cannot be compensated with the generationof new neurons, therefore the imbalance between the production of ROSand antioxidants has been implicated in several neurological disorders.Glutathione is the predominant antioxidant in the nervous system. Theglutathione content of brain cells depends strongly on the availabilityof precursors of glutathione. Alpha-lactalbumin supplements cysteine,the amino acid precursor to glutathione.

Brain serotonin levels increase under stress since the neurotransmitteris important in regulating emotional states and moods. Chronic stressand anxiety may lead to a depletion of available concentrations ofserotonin and its precursor tryptophan, causing serotonin to fall belowfunctional needs. Alpha-lactalbumin contains tryptophan and a mix ofother amino acids from a natural food protein source, and studiessuggest that dietary supplementation of alpha-lactalbumin improvescognitive performance in stress-vulnerable subjects via increased braintryptophan and serotonin activities. In one embodiment,alpha-lactalbumin synergizes the serotonergic effects of the otheringredients in the formulation, thus providing an additional mode ofaction.

S-adenosylmethionine (SAMe) is a naturally occurring compound that ispresent in tissues throughout the body. At the molecular level, SAMe isinvolved in various metabolic pathways, including transmethylation,trans sulfuration, and aminopropylation. In the body, SAMe issynthesized from an amino acid, methionine, and a triphosphatenucleotide, ATP. SAMe is in turn involved in the biosynthesis ofnumerous biological molecules, including hormones and neurotransmiters.

Administering SAMe to subjects has been found to have a variety ofsalutary effects. SAMe regulates gene expression and helps preventgenetic mutations; it maintains mitochondrial function; it participatesin phospholipid synthesis and maintains the integrity of cell membranes;and it regulates neurotransmitters such as serotonin, dopamine andepinephrine (adrenaline), and hormones such as estrogen and melatonin.SAMe is also known to inhibit neuron death following ischemia; improvethe utilization of glucose in the brain; inhibit brain edema; improvesEEG and evoked potential findings by normalizing them; and improve motorfunction, such as that impaired by stroke. SAMe has been found, forexample in meta-analyses of multiple drug studies, to enhance emotionalwell-being and is as effective as many common prescription drugs intreating depression, but with significantly fewer side effects than anyof these drugs. SAMe has also been used to treat anxiety, chronic pain,arthritis, rheumatoid fibromyalgia, Chronic Fatigue Syndrome, cognitivedifficulties associated with Alzheimer's Disease, neurovascular diseaseand neurological conditions associated with AIDS. In addition todiseases of the central and peripheral nervous system, SAMe has beenfound to improve diseases of the joints, cardiovascular system, andliver.

SAMe administration was initially considered impractical, due to theinstability of the SAMe ion during manufacturing, shipping, and storage.Eventually stable salts of SAMe were developed (such as SAMe tosylatedisulfate, the butanedisulfonate salt of SAMe, the di-para-toluenesulfonate disulfate salt of SAMe, the tri-para-toluene sulfonic acidsalt of SAMe, butanedisulphonate salt of SAMe, and disulfatep-toluensulfonate salt of SAMe). In some exemplary embodiments of thepresent invention, SAMe is administered as a commercial SAMe salt ofphytic acid.

The skilled artisan will appreciate that the composition of Magnoliaofficinalis, Phellodendron amurense, L-theanine, and whey protein mayinclude any amount of each ingredient. The ingredients may also beconcentrates, such as a whey protein concentrate. Moreover, thecomposition can be used by a human or non-human animal (e.g. mammal,avian, fish, reptilian, etc.) subjects.

In one embodiment, the composition for non-human animals may containbetween 0.5-3 mg Magnolia officinalis, 0.03-0.2 mg Phellodendronamurense, 17.0-450 mg L-theanine, and 12.0-100 mg alpha-lactalbumin.However, in another embodiment, the composition for non-human animalsmay contain between 0.01 mg-10 g Magnolia officinalis, 0.01 mg-10 gPhellodendron amurense, 0.01 mg-10 g L-theanine, and 0.01 mg-10 galpha-lactalbumin.

In one embodiment, the composition may contain at least 0.5 mg ofRELORA, at least 2.0 mg of L-theanine, at least 0.5 mg of whey protein,and at least 1.5 mg of SAMe.

In one embodiment, the composition for non-human animals such as caninesmay contain at least 6.0 mg of RELORA, at least 3.0 mg of L-theanine, atleast 1.0 mg of whey protein, and at least 3.0 mg of SAMe.

In one embodiment, the composition for non-human animals such as felinesmay contain at least 3.0 mg of RELORA, at least 2.0 mg of L-theanine, atleast 0.5 mg of whey protein, and at least 1.0 mg of SAMe.

In one embodiment, the composition for non-human animals such as equineanimals may contain at least 20.0 mg of RELORA, at least 15.0 mg ofL-theanine, at least 5.0 mg of whey protein, and at least 10.0 mg ofSAMe.

In one embodiment, the composition for humans may contain at least 20.0mg of RELORA, at least 15.0 mg of L-theanine, at least 5.0 mg of wheyprotein, and at least 10.0 mg of SAMe.

In one embodiment for companion animals such as canines or felines, thecomposition comprises 37 mg of RELORA, 17 mg L-theanine and 12 mg ofwhey protein in a single dosage form.

In one embodiment for companion animals such as canines or felines, thecomposition comprises 75 mg of RELORA, 35 mg L-theanine and 25 mg ofwhey protein in a single dosage form.

In one embodiment for companion animals such as canines or felines, thecomposition comprises 450 mg of RELORA, 205 mg L-theanine and 100 mg ofwhey protein in a single dosage form.

In one embodiment for companion animals such as canines or felines, thecomposition comprises 0.5 mg Magnolia officinalis extract, 0.03 mgPhellodendron amurense extract, 17 mg L-theanine and 12 mgalpha-lactalbumin in a single dosage form.

In one embodiment for equine animals, the composition comprises 750 mgof RELORA, 150 mg of L-theanine, 1000 mg of whey protein.

In one embodiment for equine animals, the composition comprises 1500 mgof RELORA, 300 mg of L-theanine, 1000 mg of whey protein.

One will appreciate that any combination of magnolia, phellodendron,theanine, and/or whey protein (or alpha-lactalbumin) may be combined.For instance, in one embodiment, the composition comprises magnoliaextract and alpha-lactalbumin, without phellodendron or theanine.Alternatively, the composition may include phellodendron extract andalpha-lactalbumin without magnolia or theanine. Again, any singlecomponent of magnolia, phellodendron, theanine, and/or whey protein (oralpha-lactalbumin) that provides the desired response or any combinationof multiple components can be provided. The formulation may also includevarious other flavonoids, omega-3 fatty acids, eicosapentaenoic acid(EPA) and docosahexaenoic acid (DHA), valerian, SAMe, other milkproteins, or other milk proteins (such as alpha-S1 Tryptic casein) orproducts containing milk proteins, and/or Sceletium tortuosum.

The composition can be combined in any manner and presented to the humanor non-human animal in any combined form. In one embodiment, thecomposition comprises a unit dosage form, including but not limited topharmaceutical dosage forms suitable for oral, rectal, intravenous,subcutaneous, intramuscular, transdermal, transmucosal, and topical.

In one embodiment, the composition comprises an orally administrabledosage form. Examples of orally administrable dosage forms include, butare not limited to a tablet, capsule, powder that can be dispersed in aliquid or sprinkled on food, a liquid such as a solution, suspension, oremulsion, a soft gel/chew capsule, a chewable bar, or other convenientdosage form known in the art. In some embodiments, the compositioncomprises a tablet, capsule, or soft chewable treat. The orallyadministrable dosage forms may be formulated for immediate release,extended release or delayed release. The composition may be coated oruncoated.

Example 1. Evaluation of Neurotransmitter Release Patterns inHippocampal Dialysate Samples of C57B1/6 Mice after 7 Day Treatment withan Anxiolytic Composition

Neurotransmitter release patterns in the brain were measured by aprocedure which involved inserting a probe into the hypothalamus region(microdialysis probe) of a laboratory animal and flushing the probe viadialysis with an artificial cerebrospinal fluid (CSF). This is anaccepted and common method used to obtain and measure in vivo brainlevels of neurotransmitters and other small proteins.

The fluid being collected represents the extracellular or interstitialfluid found in the hippocampal region of the brain. This area is usuallythe preferred and typical area for microdialysis analysis because anychanges measured in this area of the brain are believed to correspondwith approximately 80% of whole brain tissue. The levels ofneurotransmitters in the fluid represent a complex balance between therelease of these neurotransmitters by pre-synaptic neurons, there-uptake of these neurotransmitters back into the neurons, as well asany interactions between neurotransmitters. Therefore, the resultsindicate how compounds and their combinations affect brainneurochemistry.

For the evaluation, male C57BI mice were divided into groups (6 pergroup) and administered a variety of compounds or a placebo orally forseven days. On day 6, each mouse was anesthetized and a microdialysisprobe was inserted into the hippocampus region of the brain.Microdialysis sampling began the day after surgery, with each probebeing connected to a microperfusion pump and perfused with an artificialCSF. Samples were taken in 30 minute aliquots beginning 90 minutes priorto dosing of each compound on the 7th day. Each aliquot was thenanalyzed to determine the amount of GABA, glutamate, and serotonin(5-HT) in each sample. Analysis of the neurotransmitters was performedin two stages in an attempt to identify the chronic and acute effects ofeach compound.

In the described results, Compound A is L-theanine; Compound B is acomposition comprising an extract of Magnolia officinalis and an extractof Phellodendron amurense; Compound C is a milk whey protein comprisingalpha-lactalbumin, Compound D is SAMe; and Compound E is glutathione. Inembodiments, Compound B was provided as a commercially availablepreparation (RELORA; InterHealth Nutraceuticals, Inc., Benicia, Calif.).In embodiments, Compound C was standardized to 80% alpha-lactalbumin. Inembodiments, Compound D was provided as a commercially available SAMephytate salt. Compound A was administered at a dose of 6.66 mg/kg.Compound B was administered at a dose of 12.5 mg/kg. Compound C wasadministered at dose of 2.36 mg/kg. Compound D was administered at adose of 6.02 mg/kg. Compound E was administered at a dose of 13.6 mg/kg.Equivalent dosages for cross-species extrapolation can be calculated byone skilled in the art using conventional dose conversion methods.

Basal Neurotransmitter Output.

First, basal output levels of each neurotransmitter were measured todetermine any long-term, chronic effects of each compound on theneurotransmitters. Basal levels represent the lowest levels of eachneurotransmitter in a 24 hour cycle. These levels were determined byaveraging the levels of each neurotransmitter in the three, 30-minutealiquots prior to the administration of the 7th daily dose (Sample −90min to −60, Sample −60 minutes to −30, and Sample −30 minutes to time0). The basal levels were measured as a representation of the chroniceffects of the compounds on the levels of the neurotransmitters, i.e.measuring what should be lowest effect observed prior to a new dose. Todetermine the acute effects of these compounds, the levels of eachneurotransmitter were then analyzed in the aliquots following dosing forup to 3½ hours (210 minutes) following the 7th daily administration ofeach compound/combination.

The basal output of serotonin (5-HT), GABA, and glutamate was measuredfollowing 6 days of administration of each of the individual componentsof the described composition as well as the combination of ingredients.The mean concentration of each neurotransmitter was then compared to themean basal output measured in the placebo group using a student's t-testto determine if the two sets of data are significantly different.

With respect to serotonin, individually no single one of the evaluatedcompounds had a statistically significant effect (p-value <0.05) on thebasal output of serotonin (See FIG. 1). That is, for example,individually none of L-theanine, Relora, nor milk whey protein had anysignificant effect on the basal serotonin levels. On the other hand, thecombination of Compounds A+B+C (theanine+magnolia/phellodendron+milkwhey protein), however, did have a statistically significant effect onbasal serotonin levels (p<0.05). The combination of L-theanine and milkwhey protein (A+C) also significantly lowered basal serotonin levels.These results demonstrate an unexpected synergistic effect on brainserotonin levels of the combination.

The combinations of theanine/magnolia/phellodendron (A+B) and L-theanineand milk whey protein (A+C) produced an unexpected and synergisticincrease in GABA levels (see FIG. 2) Likewise, the combination oftheanine and SAMe (A+D) significantly increased basal GABA output.

For basal glutamate output (see FIG. 3), the combination oftheanine/magnolia/phellodendron (A+B) showed an increase. Likewise, thecombination of theanine and SAMe (A+D) significantly increased basalglutamate output.

Acute Neurotransmitter Output

Following administration of the 7^(th) dose of each compound and/orcombination, aliquots of CSF were collected in 30-minute increments upto 210 minutes following dosing. The concentration of neurotransmittersin each aliquot represents acute, shorter-term changes in the levelswhich may not be represented in the basal output results.

The results are plotted in graphs wherein the x-axis represents the timeafter administration of the 7^(th) daily dose, while the y-axisrepresents a % increase/decrease of the basal output found in the priorportion of the study. This measurement is used because the actualconcentrations of neurotransmitters can vary significantly betweensubject to subject. This method allows for a standardization of thedata, and, in essence, allows for each animal to serve as its ownindividual control. The results were then analyzed using an analysis ofvariance (ANOVA) to detect any statistically significant treatmenteffects.

Looking at the acute effects of these compounds on GABA first, nostatistically significant treatment effects were seen via ANOVA (p<0.05)for the individual compounds A, B, or C when compared to the placeboduring the test period. There was, however, a significant treatmenteffect of the combination of ingredients (A+B+C) when compared toplacebo when analyzed by ANOVA, as seen in FIG. 4. Therefore, theseresults indicate that there is a significant synergistic effect on GABAwith the combination of L-theanine and magnolia/phellodendron and milkwhey protein (A+B+C), whereas each individual component by itself has noeffect.

With respect to glutamate, neither the individual components (A, B, C)nor the combination (A+B+C) had an immediate treatment effect onglutamate levels. However, the results demonstrated a trend indicating atreatment effect by the combination (A+B+C) 120 to 210 minutes followingadministration (see FIG. 5). The skilled artisan will appreciate thatchanges in glutamate levels are one of the more difficultneurotransmitter changes to elicit.

Example 2. Evaluation of Anxiolytic Properties of the DisclosedCompositions by Behavioral Testing

Wistar rats (16 per group) were dosed either with individual components(L-theanine, magnolia/phellodendron, and milk whey protein) or severalcombinations of the three, including the three-way combination(theanine+magnolia/phellodendron+milk whey protein).Magnolia/phellodendron combination was provided as a commerciallyavailable preparation (RELORA; InterHealth Nutraceuticals, Inc.,Benicia, Calif.). Administered milk whey protein was standardized to 80%alpha-lactalbumin. The rats were dosed daily for 14 days, and then aseries of behavioral assays were performed to detect any anxiolyticeffects on the rats or sedative effects. The treatment groups were asfollows:

1: L-theanine+magnolia/phellodendron 2: L-theanine+Milk Whey Protein

3: magnolia/phellodendron+Milk Whey Protein

4: L-theanine+magnolia/phellodendron+Milk Whey Protein 5: L-theanine

6: magnolia/phellodendron

7: Milk Whey Protein

Animals were placed in the following groups:

Total Group Daily Dose Dose Dosing # of Number Treatment DoseConcentration Volume Route Animals 1 #1 50 mg/kg 10 mg/ml 5 ml/kg PO 16♂ 2 #2 24 mg/kg 4.8 mg/ml 5 ml/kg PO 16 ♂ 3 #3 42 mg/kg 8.4 mg/ml 5ml/kg PO 16 ♂ 4 #4 58 mg/kg 11.6 mg/ml 5 ml/kg PO 16 ♂ 5 #5 16 mg/kg 3.2mg/ml 5 ml/kg PO 16 ♂ 6 #6 34 mg/kg 6.8 mg/ml 5 ml/kg PO 16 ♂ 7 #7 8mg/kg 1.6 mg/ml 5 ml/kg PO 16 ♂ 8 Vehicle 0 mg/kg 0 mg/ml 5 ml/kg PO 16♂ (Sterile Water)

Elevated Plus-Maze Test

The elevated plus-maze (EPM) capitalizes on the natural aversion (traitanxiety) of rodents on brightly lit, open, and elevated areas. The EPMhas very strong predictive ability and is often used to profile thepotential anxiolytic activity of compounds. During this test, the ratsare videotaped after being placed in the elevated maze. The videos areanalyzed to determine the % of time the rats stay in the open, elevatedportions of the maze. Rats that spend more time in the open arms arepresumed to be less anxious. Table 1 below represents the average amountof time each rat spent in the open arms of the maze.

TABLE 1 Mean % Time in Treatment open arms ± SEM Vehicle 10 ± 2 1 19 ± 32 15 ± 2 3 18 ± 3 4 13 ± 3 5 13 ± 2 6 12 ± 3 7 11 ± 2

In this study, the rats administered the combination of ingredients inTreatment 4 spent, on average, 3 minutes longer in the open arms of themaze than the rats administered a placebo (see FIG. 6A), indicating ananxiolytic effect of the compounds. Similarly, the combination ofL-theanine/milk whey protein (treatment 2) spent, on average, 5 minuteslonger in the open arms of the maze than the rats administered a placebo(see FIG. 6), indicating an anxiolytic effect of the compounds. Thecombinations of L-theanine/magnolia/phellodendron (treatment 1) andmagnolia/phellodendron/milk whey protein (treatment 3) showed astatistically significant effect in post hoc pairwise comparisons (bothat p<0.05; see FIGS. 6B and 6C, respectively).

Open-Field Activity as a Measure of Sedation

Locomotor activity in an open field test is a good measure of thesedative properties of a compound. As one example, a noted side-effectof many anxiolytic compounds such as benzodiazepines is marked sedationin conjunction with administration. These products are quite effectiveanxiolytics, but patients are often saddled with the undesirable effectsof sedation.

This test is performed by placing a rat in an open, brightly litenclosure. The rats are videotaped, and the total distance the rattravels during the testing period is recorded as an indicator oflocomotor activity. Sedated rats will travel less, while active ratswill travel farther total distances.

TABLE 2 Mean Distance Treatment (cm) ± SEM Vehicle 9062 ± 597 1 11735 ±447   2^(a) 10887 ± 1055 3 11068 ± 814  4 9833 ± 789 5 9971 ± 652 610224 ± 635  7 10191 ± 643  ^(a)One rat removed from analysis due toinaccurate video tracking

The results presented in Table 2 and FIG. 7A indicate that none of thecompounds/combinations administered appear to have a sedative effect onthe locomotor activity of the rats. All rats were more active than theplacebo, indicating an enhanced desire to explore and investigate, andindeed post hoc pairwise comparisons indicated a trend towards increasedlocomotor activity, particularly for Treatment 1 [030)=3.58, p<0.01;t-test] and Treatment 3 [030)=1.99, p=0.06; t-test] (see FIGS. 7B and7C). Compounds which have an anxiolytic effect without sedation arethought to be extremely desirable candidates for treatment of anxiety.

Accordingly, by the foregoing results it will be appreciated that aneffective composition suitable for oral administration is provided whichin various embodiments affects brain neurochemistry as shown byalterations in release patterns of various neurotransmitters. In turn,various behavioral evaluations demonstrated an anxiolytic effect ofvarious embodiments of the described compositions. While individualcompounds of the described compositions had no effect on brainneurochemistry or behavior, surprisingly a synergistic effect of thecompositions was seen.

The foregoing has been presented for purposes of illustration anddescription. It is not intended to be exhaustive or to limit theembodiments to the precise form disclosed. Obvious modifications andvariations are possible in light of the above teachings. The embodimentsdescribed above were chosen to provide the best application to therebyenable one of ordinary skill in the art to utilize the disclosedinventions in various embodiments and with various modifications as aresuited to the particular use contemplated. All such modifications andvariations are within the scope of the appended claims when interpretedin accordance with the breadth to which they are fairly, legally andequitably entitled.

What is claimed:
 1. An anxiolytic composition, comprising a synergisticcombination of L-theanine, a whey protein, an extract of magnolia, andan extract of phellodendron.
 2. The anxiolytic composition of claim 1,wherein the whey protein comprises alpha-lactalbumin.
 3. The anxiolyticcomposition of claim 1, wherein the synergistic combination provides ananxiolytic change in a release pattern of one or more neurotransmitters.4. The anxiolytic composition of claim 3, wherein the brainneurotransmitters are selected from the group consisting ofγ-aminobutyric acid (GABA) and serotonin.
 5. The anxiolytic compositionof claim 1, formulated for oral administration to a mammal.
 6. Theanxiolytic composition of claim 5, wherein the mammal is selected fromthe group consisting of a human, a companion animal, and an equineanimal.
 7. The anxiolytic composition of claim 1, wherein thesynergistic combination provides an anxiolytic change in a releasepattern of one or more neurotransmitters.
 8. The anxiolytic compositionof claim 7, wherein the brain neurotransmitters are selected from thegroup consisting of γ-aminobutyric acid (GABA) and serotonin.
 9. Theanxiolytic composition of claim 1, wherein the synergistic combinationcomprises RELORA.
 10. A method for reducing, ameliorating, or treatingsymptoms of anxiety, comprising administration of a synergisticcombination of L-theanine, a whey protein, an extract of magnolia, andan extract of phellodendron to a mammal.
 11. The method of claim 10,further including providing the whey protein as a whey proteinconcentrate comprising alpha-lactalbumin.
 12. The method of claim 10,including orally administering the synergistic combination in an amountsufficient to provide an anxiolytic change in a release pattern of oneor more neurotransmitters.
 13. The method of claim 12, wherein the brainneurotransmitters are selected from the group consisting ofγ-aminobutyric acid (GABA) and serotonin.
 14. The method of claim 10,wherein the mammal is a canine or feline.
 15. The method of claim 10,wherein the mammal is an equine.
 16. An anxiolytic composition,comprising a synergistic combination of L-theanine, a whey protein, andRELORA.
 17. The anxiolytic composition of claim 16, wherein the wheyprotein is a whey protein comprising alpha-lactalbumin.
 18. Theanxiolytic composition of claim 16, wherein the whey protein is a wheyprotein concentrate.
 19. The anxiolytic composition of claim 16, whereinthe composition comprises at least 2.0 mg of L-theanine, at least 0.5 mgof whey protein, and at least 0.5 mg of RELORA.
 20. The anxiolyticcomposition of claim 16, wherein the composition comprises at least 2.0mg of L-theanine, at least 0.5 mg of whey protein, and at least 3.0 mgof RELORA.